Our long-term objective is to understand the function and mechanism of microbial multidrug resistance pumps (MDRs). The main goal of this project is to identify natural substrates and inhibitors of microbial MDRs. According to our findings, berberine alkaloids are natural MDR substrates. Plants making berberine alkaloids also produce at least two MDR inhibitors (5'-methoxyhydnocarpin and pheophorbide) that act in synergy with berberine. We will use this model for an expanded search of new MDR substrates among plants producing antimicrobials, and for novel MDR inhibitors that are likely to accompany them. Our main focus will be on plants that make non-cationic antimicrobials that can be extruded by broad-specificity MDRs of Gram negative bacteria and yeast. This is a collaborative project that brings together the expertise of the PI in microbial multidrug resistance and the expertise of the co-PI in natural products chemistry. The overall project will flow from identifying antimicrobials that are MDR substrates, to finding MDR inhibitors in plants that produce these antimicrobials, to characterizing the interaction of these substances with microbial MDRs. The Specific Aims of this proposal are: 1. Identifying MDR substrates among plant antimicrobials. Plant antimicrobials will be screened with a panel of Gram positive bacteria, Gram negative bacteria and yeast in search of MDR substrates. We will screen a large collection of substances, representing the main types of antimicrobials (alkaloids, flavones, quinones, phenols) coming from a variety of unrelated plants. Antimicrobials that are more active against strains lacking MDRs are likely MDR substrates. 2. Searching for MDR inhibitors. Once MDR substrates are identified, the producing plant will be used to isolate MDR inhibitors. A bioassay-driven purification will be used, based on detecting inhibition of cell growth by a test extract in the presence of a sub-inhibitory concentration of an MDR substrate. The structure of newly identified MDR inhibitors will be determined. 3. Characterizing MDR inhibitors. (a) Spectrum of activity regarding the MDR family, and the type of organism will be determined. (b) Interaction of the inhibitor with MDRs will be studied using a transport assay with microbial cells. (c) SAR of MDR inhibitors will be performed. Relation to human health. MDR inhibitors are a new type of plant defense compounds that we are exploring. By potentiating other antimicrobials, MDR inhibitors may provide the key for developing plant antimicrobials into new antibiotics. MDR inhibitors will potentiate the action of conventional antibiotics, aiding eradication of multidrug resistant human pathogens.